In a normal human cell, about 2 meters of DNA are folded and packaged into a nucleus less than 10 microns in diameter. Much of this compaction is performed by wrapping the DNA around nucleosomes, which are complexes composed of histone proteins. Compaction and packaging into the nucleus provides a level of protection for fragile strands of DNA. However, despite the compaction of DNA into chromosomes, double-stranded breaks in DNA are common.
DNA double-stranded breaks jeopardize a chromosome's physical integrity essential for its correct segregation during mitosis and meiosis as well as its informational redundancy critical for maintaining accurate encoding of cellular components. Given that DNA double-stranded breaks have the potential to cause a great deal of damage to the cell, it is not surprising that multiple cellular mechanisms exist for dealing with this serious lesion, including non-homologous end joining, homologous recombination, and apoptosis. On the other hand, DNA double-strand cleavages are necessary in several important cellular processes, including recombination during meiosis and mitosis, V(D)J recombination during immune system development, and mating type switching in S. cerevisiae. Aside from natural processes, DNA double-stranded breaks also appear following exposure to radiation, magnetic fields, toxins, mutagenic chemicals, various medications, and the like.
While much is known about the causes and actual rejoining of DNA double-stranded breaks, much less is known about how these breaks are initially recognized. Elucidation of an organism's initial response mechanisms to double-stranded breaks would provide a means to detect DNA double-stranded breaks. Currently, DNA breaks are detected using pulsed-field electrophoresis, filter elution, sucrose gradient sedimentation, single cell gel electrophoresis or the TdT-mediated fluorescein-dUTP nick end labeling (TUNEL) assay. The former three methods are useful for only gross measurements of DNA damage in a large sample.
Single cell gel electrophoresis, also known as the comet assay, capitalizes on the movement of fragmented DNA out of a cell body into the agarose gel toward the anode during electrophoresis. The resulting shape resembles a comet, the cell body being the comet head and the fragmented DNA being the comet tail. The comet assay, frequently used in the study of apoptosis, is an improvement over the aforementioned techniques in that it allows measurement of DNA cleavage in an individual cell. However, this technique requires a great deal of time and effort to study DNA breaks in larger samples and lacks sensitivity.
The TUNEL assay employs terminal deoxynucleotidyl transferase to incorporate modified nucleotides onto free 3′-hydroxyl ends of DNA fragments. In most cases, DNA breaks are visualized by differential staining of intact and fragmented DNA. However, the TUNEL assay, as well as the comet assay, require many hundreds of DNA double-stranded breaks to yield a positive signal.
In that only a few double-stranded breaks can result in phenotypic change and 40 double-stranded breaks result in cell death, the currently available assays clearly do not have the sensitivity to be useful in all situations, particularly with non-lethal amounts of breaks. In view of the above, there exists a need for a sensitive means of determining DNA double-stranded breaks. It is an object of the present invention to provide such a means. This and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.